Hindbrain Ghrelin Receptor Signaling Is Sufficient to
Maintain Fasting Glucose
Michael M. Scott1,2, Mario Perello1¤, Jen-Chieh Chuang1, Ichiro Sakata1, Laurent Gautron1,
Charlotte E. Lee1, Danielle Lauzon1, Joel K. Elmquist1,3,4,5*., Jeffrey M. Zigman1,3,4*.
1 Division of Hypothalamic Research, Department of Internal Medicine, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of
America, 2 Department of Pharmacology, University of Virginia, Charlottesville, Virginia, United States of America, 3 Division of Endocrinology & Metabolism, Department
of Internal Medicine, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America, 4 Department of Psychiatry, The University of
Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America, 5 Department of Pharmacology, The University of Texas Southwestern Medical
Center at Dallas, Dallas, Texas, United States of America
Abstract
The neuronal coordination of metabolic homeostasis requires the integration of hormonal signals with multiple interrelated
central neuronal circuits to produce appropriate levels of food intake, energy expenditure and fuel availability. Ghrelin, a
peripherally produced peptide hormone, circulates at high concentrations during nutrient scarcity. Ghrelin promotes food
intake, an action lost in ghrelin receptor null mice and also helps maintain fasting blood glucose levels, ensuring an
adequate supply of nutrients to the central nervous system. To better understand mechanisms of ghrelin action, we have
examined the roles of ghrelin receptor (GHSR) expression in the mouse hindbrain. Notably, selective hindbrain ghrelin
receptor expression was not sufficient to restore ghrelin-stimulated food intake. In contrast, the lowered fasting blood
glucose levels observed in ghrelin receptor-deficient mice were returned to wild-type levels by selective re-expression of the
ghrelin receptor in the hindbrain. Our results demonstrate the distributed nature of the neurons mediating ghrelin action.
Citation: Scott MM, Perello M, Chuang J-C, Sakata I, Gautron L, et al. (2012) Hindbrain Ghrelin Receptor Signaling Is Sufficient to Maintain Fasting Glucose. PLoS
ONE 7(8): e44089. doi:10.1371/journal.pone.0044089
Editor: Raul M. Luque, University of Cordoba, Spain
Received June 12, 2012; Accepted August 1, 2012; Published August 31, 2012
Copyright: ß 2012 Scott et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funds used to conduct this study were provided by the National Institutes of Health, specifically, NIDA, NIMH, and NIDDK. The relevant grants are listed
below: 5K99DA024719-02 to M.S., 1R01DA024680, 1R01MH085298, and 1K08DK068069 to J.M.Z., and R01DK071320 and RL1DK081185 to J.K.E. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] (JKE); [email protected] (JMZ)
. These authors contributed equally to this work.
¤ Current address: Laboratory of Neurophysiology, Multidisciplinary Institute of Cell Biology, La Plata, Buenos Aires, Argentina
ing the medial basal hypothalamus, hindbrain, and midbrain,
including sites already known to mediate food intake and glucose
homeostasis [14]. Also, ghrelin can induce c-fos expression in
many of these sites, suggesting its ability to activate their resident
neurons, the end result of which could be changes to behaviors
and processes controlling food intake and blood glucose [6,15].
For instance, direct injection of ghrelin into the hypothalamic
arcuate nucleus stimulates food intake while interference with
signaling by arcuate AgRP/NPY neurons has the opposite effect
[6,16]. Ghrelin injection into the dorsal vagal complex of the
hindbrain also induces feeding and recapitulates the induction of
c-fos produced by intracerebroventricular ghrelin injection [17].
Increased food intake also occurs upon ghrelin injection into the
ventral tegmental area (VTA) of the midbrain [18,19], an effect
lost in the ghrelin receptor null animals. The relative contribution
of each of these central nuclei expressing GHSRs in the control of
ghrelin-induced feeding, however, is debatable.
In this report, we describe the use of a Phox2b-Cre
recombinase-expressing mouse line that permits the selective
expression of GHSR in specific hindbrain nuclei implicated in the
control of food intake and glucose homeostasis [5]. Outside of the
hindbrain, the mouse remains null for the receptor, allowing us to
test the sufficiency of ghrelin signaling in this area to modulate
Introduction
Ghrelin is a peptide hormone secreted predominantly by a
sparsely-distributed group of endocrine cells in the gastrointestinal
mucosa [1]. Ghrelin was originally described as a potent growth
hormone releasing signal, acting through the G-protein coupled
receptor GHSR in the brain [1]. Studies since have revealed a
wider role for ghrelin in numerous metabolism-related processes.
Ghrelin is hypothesized to act as a meal initiator signal and fuel
storage gauge, secreted at times of low nutrient availability [2,3].
Not only does ghrelin potently stimulate food intake [4,5,6], but
also the manifestation of various food-reward behaviors depends
on ghrelin action in the central nervous system (CNS) [7,8].
Ghrelin also raises blood glucose, which is particularly relevant
during severe caloric restriction when it prevents marked
hypoglycemia and death [9,10,11,12], though this action of
ghrelin is likely dependent on other yet to be identified genetic and
environmental modifiers [13].
The sites of ghrelin action in the coordination of food intake and
glucose homeostasis have yet to be definitively identified, but
undoubtedly include direct action on one or more of several CNS
sites implicated in the control of nutrient intake and blood glucose.
For example, GHSRs are expressed within many nuclei compris-
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Hindbrain Ghrelin Receptors Affect Fasting Glucose
both feeding and glucose levels. Using this approach, we
demonstrate that hindbrain ghrelin signaling is not sufficient to
mediate the effects of ghrelin on acute food intake but is sufficient
to normalize blood glucose following a fast.
Data and Statistical Analyses
Brain sections were analyzed with a Zeiss Axioplan light
microscope. Data are presented as average +/2 SEM. Adobe
Photoshop 7.0 was used to adjust only sharpness, brightness, and
contrast as well as to combine selected images into plates.
Comparisons of food intake following ghrelin or saline injection
and of fasting glucose levels were performed using a 1-way
ANOVA and Tukey’s post-hoc test.
Materials and Methods
Animal Husbandry
All procedures were conducted in accordance with the UTSW
Institutional Animal Care and Use Committee guidelines and
those of AAALAC. Furthermore, the UT Southwestern IACUC
committee specifically approved the work reported in this current
study. Mice were housed in a pathogen-free facility on a 12h light/
dark cycle with ad libitum access to food and water unless specified
otherwise. Male mice were used for all experiments. GHSR-null
mice, which contain a loxP-flanked transcriptional blocking
cassette within the endogenous GHSR alleles [5], and Phox2bCre mice [20] were both on a pure C57Bl/6J genetic background.
Phox2b-Cre mice were mated to heterozygous GHSR-null
animals, and then offspring hemizigous for Phox2b-Cre and
heterozygous for the GHSR-null allele were crossed with mice
heterozygous for the GHSR-null allele to produce study animals
(wild-type, GHSR-null, GHSR-null/Phox2b). Mice hemizygous
for Phox2b-Cre and wild-type for the GHSR allele showed no
obvious phenotypic differences from wild-type animals [our
unpublished observations and Scott et al. [20]], and thus, wildtype animals were used as controls in all reported experiments.
Results
Phox2b-Cre-mediated Reactivation of GHSR Expression
To test the sufficiency of hindbrain ghrelin receptor expression
in the mediation of ghrelin-stimulated feeding, we used a mouse
model system that is conditionally null for GHSR (GHSR-null).
These mice contain a loxP-flanked transcriptional blocking
cassette inserted into intronic DNA upstream of the GHSR
translational start codon, thus blocking GHSR expression [5,7].
To re-express GHSR selectively in the hindbrain, we crossed the
GHSR-null mice to a line of mice expressing Cre recombinase
from the Phox2b locus of a bacterial artificial chromosome. These
Phox2b-Cre mice express Cre selectively in the hindbrain, within
brachial and visceral motor neurons and cells of the nucleus of the
solitary tract (NTS) and area postrema [20,22]. In characterizing
the GHSR-null/Phox2b mice (which contain two GHSR-null
alleles and Phox2b-Cre), we determined that GHSR was
expressed in all hindbrain nuclei that had previously shown
GHSR expression [14], at levels that appeared to recapitulate that
of wild-type mice (Figure 1). In particular, just as was observed in
wild-type mice, within GHSR-null/Phox2b mice, GHSR mRNA
expression was observed by ISHH in all three components of the
dorsal vagal complex [including the NTS, dorsomotor nucleus of
the vagus nerve (DMV), and area postrema (AP)], nucleus
ambiguus (Amb) and facial motor nucleus (nVII). Expression
was absent from all other areas of the brain (including the arcuate
nucleus) of GHSR-null/Phox2b mice (figure 1 and data not
shown), demonstrating the desired and expected restriction of
GHSR expression to the hindbrain.
In Situ Hybridization Histochemistry (ISHH)
The extent of GHSR expression resulting from Phox2b-Credependent removal of the transcriptional blocking cassette as
compared to that observed in wild-type mice was determined
using single-label free-floating ISHH (n = 4), as described previously [7,21].
Ghrelin-Stimulated Food Intake
Six- to eight-week-old male mice were individually housed for 7
days prior to experimentation. Three days prior to experimentation, food was removed and replaced with 2 pellets of mouse chow
placed in a petri dish on the cage floor. Mice were acclimated to
the handling protocol for 3 days prior to s.c. injection of either
saline vehicle or 2 mg/g mouse acyl-ghrelin (Pi Proteomics,
Huntsville, Al). At 11 am on the test day, one chow pellet was
weighed and placed in a petri dish on the cage floor followed by
injection of saline into one half of the cohort and ghrelin into the
other. Chow intake was followed for 3 hr. After experimentation,
mice were singly housed for 7 days and the experiment repeated.
Mice that received ghrelin during the first experiment received
saline during the second trial and vice versa.
Induction of c-fos by Ghrelin in Mice with Hindbrainselective GHSR Expression
Multiple studies have suggested that hindbrain neurons exhibit
c-fos expression in response to exogenous ghrelin administration –
both centrally (into the 4th ventricle or directly into the hindbrain)
and peripherally (including intravenously or intraperitoneally)
[23,24,25,26,27,28]. It also is known that hindbrain delivery of
ghrelin via the 4th ventricle does not induce c-fos in the arcuate
nucleus or paraventricular nucleus as it does when administered
into the forebrain ventricles or directly into those nuclei,
suggesting the presence of partially independent forebrain and
hindbrain circuits that respond to ghrelin [23,25,29]. However, it
has not been determined definitively whether the induction of c-fos
in the hindbrain by naturally-produced ghrelin in the periphery is
the result of direct ghrelin action at the hindbrain or the result of
indirect ghrelin action outside the hindbrain.
As an initial test of the role of hindbrain GHSR receptor
expression on the sensing of ghrelin produced in the periphery, we
examined the induction of brain c-fos expression in response to
administered ghrelin. A 2 mg/g BW subcutaneous dose of ghrelin
was chosen because it can acutely induce food intake and certain
antidepressant-like and food-reward behaviors and also can
acutely elevate blood glucose [8,12]. In wild-type mice, we
observed significant c-fos expression in the hindbrain (AP and
NTS) and hypothalamus (arcuate nucleus and paraventricular
Glucose Measurements
Fed and fasted (18 hr) glucose levels were monitored between
12–3 pm using a OneTouch Ultra glucometer and testing strips
(LifeScan, Inc. Milpitas, CA). Blood was obtained from tail veins
nicked using a disposable razor blade. For the fasting measurements, food was removed just prior to lights off (6 pm) the day
preceding measurement of glucose.
c-fos Expression Analysis
Mice were injected with acyl-ghrelin, as described above. Mice
were subsequently transcardially perfused with formalin, and
sectioned brains were stained using anti-c-fos antisera, as detailed
previously [21].
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Hindbrain Ghrelin Receptors Affect Fasting Glucose
Figure 1. GHSR mRNA expression is restored selectively in the hindbrain in GHSR-null/Phox2b mice. Coronal sections of a
representative wild-type mouse brain demonstrating expression in the nucleus of the solitary tract, area postrema and vagus motor nerve (A, NTS, AP,
DMV), nucleus ambiguus (B, Amb), 7th nerve (C, nVII) and the arcuate nucleus of the hypothalamus (D, ARH). Coronal sections of a representative
GHSR-null/Phox2b mouse brain demonstrating re-activated GHSR expression in all hindbrain nuclei (E-G) but not in the ARH (H).
doi:10.1371/journal.pone.0044089.g001
resulting from ghrelin administration. We tested this hypothesis by
examining ghrelin-stimulated food intake in GHSR-null/Phox2b
mice and their wild-type and GHSR-null control littermates. As
mentioned, subcutaneous ghrelin at the 2 mg/g BW dose, as well
as at lower doses, potently stimulates food intake over a two-hour
period in wild-type mice [7,8]. Thus, as expected, feeding was
observed in wild-type mice during the first 2-hr period following
subcutaneous ghrelin administration when compared to saline
injection (Figure 3A). No effect of ghrelin was observed in the
GHSR-null/Phox2b mice, as indicated by food intake after
ghrelin injection that was identical to that of saline-injected wildtype animals and ghrelin–injected GHSR-null animals (Figure 3A).
Thus, we conclude that hindbrain expression of GHSR is not
nucleus) (Figure 2). However, ghrelin administration failed to
induce c-fos expression in GHSR-null/Phox2b mice above that of
the basal levels observed in saline-injected wild-type mice or
ghrelin-injected GHSR-null animals, indicating that hindbrain
GHSR expression was not sufficient to mediate the shift in gene
expression induced by the injection of ghrelin (Figure 2). The
induction of hindbrain c-fos in wild-type mice, therefore, must be
an indirect result of the actions of ghrelin outside of the hindbrain.
Ghrelin-stimulated Food Intake is not Rescued by
Hindbrain Expression of GHSR
We next addressed whether expression of GHSR in the
hindbrain was sufficient to mediate an increase in food intake
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Hindbrain Ghrelin Receptors Affect Fasting Glucose
Figure 2. Phox2b-Cre-mediated GHSR re-expression does not restore CNS c-fos induction. Ghrelin (2 mg/g s.c.) induces robust c-fos
expression in the arcuate nucleus (A) and the paraventricular nucleus (B) of the hypothalamus and in the area postrema and nucleus of the solitary
tract of the hindbrain (C) in wild-type mice. Saline does not induce c-fos in the brains of wild-type mice (D-F). Ghrelin administration fails to induce cfos expression in GHSR-null (G-I) and GHSR-null/Phox2b mice (J2L). (n = 3) 3v, third ventricle. cc, central canal.
doi:10.1371/journal.pone.0044089.g002
with increased insulin sensitivity and reduced circulating glucagon
[5,7,30,31]. The fall in blood glucose observed upon interference
with ghrelin signaling is exacerbated even further upon depletion
of fat stores, as occurs with prolongation and intensification of the
caloric restriction [9,11], an action clearly modified by other
genetic and environmental influences [13]. Thus, here we tested
whether the hindbrain expression of GHSR was sufficient to
rescue fasting glucose levels.
sufficient to drive the feeding response to subcutaneouslyadministered ghrelin.
Fasting Hypoglycemia is Rescued in Mice with GHSR
Expression Limited to the Hindbrain
Several studies have demonstrated that ghrelin is required to
maintain glucose homeostasis during fasting exists. For example,
GHSR deletion lowers fasting blood glucose in mice exposed to an
overnight fast; this greater fall in blood glucose has been associated
Figure 3. Phox2b-Cre-mediated GHSR re-expression fails to normalize ghrelin-stimulated feeding but restores fasting glucose
levels. Ghrelin (2 mg/g s.c.) potently induces 2-hr food intake when compared to saline injection in wild-type but not in GHSR-null mice or in GHSR
null/Phox2b cre mice. (n = 7, * = P,0.05 One-way ANOVA with Tukey’s post-hoc test) (A). Hindbrain-selective GHSR expression restores fasting blood
glucose to that of wild type. (n = 25, * = P,0.05 1-way ANOVA with Tukey’s post-hoc test C).
doi:10.1371/journal.pone.0044089.g003
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Hindbrain Ghrelin Receptors Affect Fasting Glucose
Although glucose levels were identical in ad libitum-fed wild-type,
GHSR-null and GHSR-null/Phox2b mice (Figure 3B), following a
fast, GHSR-null mice exhibited significant hypoglycemia compared to wild-type mice, as expected (Figure 3C). Notably,
selective GHSR expression in the hindbrain was sufficient to
normalize this relative hypoglycemia, as fasting glucose levels in
the GHSR-null/Phox2b mice were significantly different from
those in GHSR-null mice n = 25) p,0.05) and not significantly
different from those in wild-type mice (Figure 3C).
hindbrain neurons is not sufficient on its own to mediate these
acute orexigenic effects of ghrelin. Conversely, hindbrain ghrelin
signaling does contribute to the maintenance of fasting glucose
levels, as evidenced by the recapitulation of the usual (e.g.
observed in wild-type mice) blood glucose response to an overnight
fast by hindbrain-selective GHSR expression.
These data complement our prior studies investigating the
effects of tyrosine hydroxylase-Cre-driven GHSR expression, in
which GHSR expression occurs selectively in catecholaminergic
(predominantly dopaminergic) neurons, such as those in the VTA
(Chuang et al., 2011a). Notably, and unlike with Phox2b-Credriven hindbrain GHSR expression, catecholaminergic GHSR
expression was sufficient to partially rescue ghrelin-stimulated
acute food intake, while also fully restoring the ability of
administered ghrelin and chronic stress to modulate food reward
(Chuang et al., 2011a). Also unlike with the hindbrain-selective
GHSR expression observed here, fasting blood glucose levels were
not rescued by selective GHSR expression in catecholaminergic
cells (Chuang et al., 2011a).
Regarding the involvement of ghrelin with the control of blood
glucose, it most likely is the case that ghrelin’s full effect includes
direct actions not only on the hindbrain, but also on other CNS
sites and peripheral organs that affect glucose homeostasis. For
instance, ghrelin has the capacity to directly bind to GHSRs on
pancreatic alpha cells and beta cells, leading to stimulation of
glucagon release and inhibition of insulin release, respectively,
both of which would tend to raise blood glucose levels [37]. The
ability of ghrelin to potently stimulate growth hormone (GH)
secretion is potentially important in blood glucose homeostasis, as
evidenced by the marked hypoglycemic and insufficient GH
responses to severe caloric restriction in mice lacking ghrelin, and
the correction of the marked hypoglycemia by pharmacologic
preservation of the usual GH response [9,11]. Interestingly, a
recent report demonstrates that this role of ghrelin may be
dependent on several modulating factors [13], complicating the
investigation of ghrelin function. Thus, further studies will be
required to determine in just what settings and in just what
manner GHSR-expressing hindbrain neurons coordinate with
other directly ghrelin-responsive neurons, pancreatic islet cells and
pituitary cells to modulate blood glucose. Additional studies also
will be needed to more extensively describe the integrated
neuronal circuitry through which ghrelin contributes to food
intake as well as to other more complex eating behaviors,
including other CNS sites that are sufficient for and/or required
for ghrelin’s orexigenic actions. Notwithstanding these as-of-yet
unanswered questions, the data here does add significantly to our
understanding of the actions of ghrelin, highlighting the distributed nature of ghrelin signaling within the CNS.
Discussion
In this study, we have employed a novel genetically-engineered
mouse model with ghrelin receptor (GHSR) expression limited to
the hindbrain to determine if such site-selective, hindbrain GHSR
expression is sufficient to mediate ghrelin’s actions on food intake
and blood glucose. With respect to intake of freely-available food,
hindbrain GHSR expression was not sufficient to permit the
characteristic orexigenic response to subcutaneous ghrelin administration observed in wild-type animals. With respect to the
modulation of glucose homeostasis, hindbrain GHSR expression
was sufficient to defend against the exacerbated fasting-induced
fall in blood glucose that is otherwise observed in mice with global
GHSR deficiency. Interestingly, although subcutaneous ghrelin
administration induces c-fos in the hindbrain of wild-type animals,
such does not occur when GHSR expression is limited to the
hindbrain. Thus, hindbrain c-fos induction seems dispensable for
ghrelin-dependent modulation of fasting glucose levels. These data
help clarify the relevant sites of ghrelin receptor action in the brain
in the modulation of food intake and blood glucose.
Several previous studies have implicated both the hypothalamus
and the hindbrain as important CNS regions mediating ghrelin’s
orexigenic actions. Among the many studies focusing on the
hypothalamus, genetic ablation of both neuropeptide Y and
agouti-related protein, which are normally co-expressed in a group
of GHSR-expressing, arcuate neurons, was shown to completely
abolish the acute orexigenic action of intraperitoneally-administered ghrelin [32]. Similarly, preventing release of the inhibitory
neurotransmitter GABA from these arcuate NPY/AgRP neurons
markedly attenuates acute food intake in response to intraperitoneal ghrelin [33]. In studies focusing on the hindbrain, direct
microinjection of ghrelin into the dorsal vagal complex (which
includes GHSR-containing neurons in the AP, NTS and dorsal
motor nucleus of the vagus) was shown to stimulate food intake, at
a dose lower than the lowest effective dose shown to induce food
intake upon microinjection into the arcuate nucleus [16,17].
Delivery of ghrelin to the dorsal vagal complex and the rest of the
caudal brainstem, via injection into the fourth ventricle, also
acutely increases food intake, number of meals, and speed of first
meal onset; such is comparable to those changes elicited by ghrelin
infusion into the third ventricle, in which ghrelin is exposed
additionally to the arcuate nucleus [17]. Interestingly, total
subdiaphragmatic vagotomy blocks the orexigenic actions of
ghrelin upon its peripheral administration, and such is thought
to occur independently of vagal afferent signaling [34,35,36].
While these latter studies indicate that GHSR-containing
hindbrain neurons have the capacity to mediate ghrelin-stimulated
food intake and that intact vagal signaling may be required for
ghrelin’s overall acute effects on food intake, the data here
demonstrates that direct sensing of ghrelin by GHSR-expressing
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Acknowledgments
We thank Sherri Osborne-Lawrence for technical assistance. We thank
Laura Brule, Mi Kim and Linh-An Cao from the Mouse Metabolic
Phenotyping Core at UT Southwestern Medical Center.
Author Contributions
Conceived and designed the experiments: MS MP JZ JE. Performed the
experiments: MS MP J-CC IS LG CL DL. Analyzed the data: MS MP JZ
JE. Wrote the paper: MS MP JZ JE.
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August 2012 | Volume 7 | Issue 8 | e44089